e. coli codon-optimized version of the trkin cds  (GenScript corporation)

Journal: Genes & Development

Article Title: Distinct kinesin motors drive two types of maize neocentromeres

doi: 10.1101/gad.340679.120

Figure Lengend Snippet: Trkin and its divergence from other kinesin-14s. ( A ) Comparison of Ab10-I-MMR and normal chromosome 10 from B73 (v5 assembly). Regions of shared, inverted synteny between N10 and Ab10 are highlighted in gray. The ∼130-kb Trkin gene is expanded above Ab10. The Kindr complex, highlighted in purple, contains nine Kindr genes in tandem. ( B ) Alignment of the Trkin gene and selected orthologs. The black boxes show exon boundaries. Predicted protein domains are also highlighted, including coiled-coil (cyan), kinesin-14 motor (yellow), and nuclear localization signal (red). ( C ) Phylogeny of Trkin and related kinesin-14 motor domains. Bootstrap values are shown at nodes.

Article Snippet: An E. coli codon-optimized version of the Trkin CDS was synthesized by Genscript and cloned into the expression vector pET-17b (Novagen) modified to include N-terminal 6xHis and GFP tags for affinity protein purification.

Techniques: Comparison

Journal: Genes & Development

Article Title: Distinct kinesin motors drive two types of maize neocentromeres

doi: 10.1101/gad.340679.120

Figure Lengend Snippet: Conservation and functionality of the TRKIN motor. ( A ) Alignment of the TRKIN motor domain and selected orthologs. Percent identity of each protein to ZmDV1 is indicated at the left . Sequence matches (in gray boxes), deletions (in black lines), and substitutions (in RasMol colors) are shown relative to the consensus of all proteins in the alignment. The position of the loop-8 motif is highlighted in blue. ( B ) Schematic of the microtubule-gliding assay. His tag antibodies (blue) adhere to the microscope slide surface and immobilize GFP-TRKIN proteins. Motor activity moves the polarity-marked microtubules along the slide. ( C ) Representative images showing that surface-immobilized GFP-TRKIN causes polarity-marked microtubules to move with the bright plus ends leading; yellow and white arrowheads indicate the plus ends of two different microtubules.

Article Snippet: An E. coli codon-optimized version of the Trkin CDS was synthesized by Genscript and cloned into the expression vector pET-17b (Novagen) modified to include N-terminal 6xHis and GFP tags for affinity protein purification.

Techniques: Sequencing, Gliding Assay, Microscopy, Activity Assay

Journal: Genes & Development

Article Title: Distinct kinesin motors drive two types of maize neocentromeres

doi: 10.1101/gad.340679.120

Figure Lengend Snippet: TRKIN localization and presence/absence in different Ab10 types. ( A ) Visualization of TRKIN during anaphase II by structured illumination microscopy. TRKIN protein immunolocalization (green) and TR-1 (red) and knob180 (cyan) sequences labeled by FISH in a line homozygous for Trkin but lacking Kindr . The image is representative of results obtained from eight different plants. Three-dimensional visualization of this cell is in Supplemental Movie S1 . ( B ) Localization of TRKIN (magenta) and tubulin (green) in Ab10-I-MMR and Ab10-II-MMR male meiocytes during anaphase II. Note the absence of specific staining in the Ab10-II-MMR line. The images shown are representative of results obtained from four (Ab10-I) and three (Ab10-II) plants. ( C ) Distribution of mRNA-seq reads from various libraries aligned to the Trkin transcript. The predicted TRKIN protein and coding exons are shown, along with the location of the peptide used to prepare antibodies (blue) for reference. Track colors reference cytologically distinguishable haplotypes: Ab10-I (green), Ab10-II (purple), Ab10-III (orange), K10L2 (red), and N10 (blue). Vertical black lines indicate mismatched bases. The Y -axes are scaled relative to the read count in the various samples and should not be interpreted as absolute values. ( D ) Correlation between Trkin and TR-1 neocentromeres in different genotypes. Merged FISH images show CentC (yellow), knob180 (green), and TR-1 (red) during anaphase II. Knob positions are denoted by colored arrows. TR-1 neocentromeres generally move faster than knob180 neocentromeres, and neocentromeres of both types arrive at the poles before centromeres. The images are representative of results obtained from analyzing three plants for each genotype. Scale bars, 10 µm.

Article Snippet: An E. coli codon-optimized version of the Trkin CDS was synthesized by Genscript and cloned into the expression vector pET-17b (Novagen) modified to include N-terminal 6xHis and GFP tags for affinity protein purification.

Techniques: Microscopy, Labeling, Staining

Journal: Genes & Development

Article Title: Distinct kinesin motors drive two types of maize neocentromeres

doi: 10.1101/gad.340679.120

Figure Lengend Snippet: Immunolocalization of TRKIN and KINDR in male meiosis. TRKIN and KINDR are shown in magenta and tubulin is shown in green. The stages of meiosis are indicated at left. The images shown are representative of data collected from eight plants. The TRKIN staining at spindle poles in prometaphase I is background staining and was not observed in all images. Scale bars, 10 µm.

Article Snippet: An E. coli codon-optimized version of the Trkin CDS was synthesized by Genscript and cloned into the expression vector pET-17b (Novagen) modified to include N-terminal 6xHis and GFP tags for affinity protein purification.

Techniques: Staining

Journal: Genes & Development

Article Title: Distinct kinesin motors drive two types of maize neocentromeres

doi: 10.1101/gad.340679.120

Figure Lengend Snippet: Possible TR-1 neocentromere functions. ( A ) Meiosis I directional switching. ( Left ) During meiosis I, sister chromatids remain attached along their entire lengths by cohesin ( ; ). Because they are physically attached, the knobs on sister chromatids are likely to migrate toward one pole, creating a knob–centromere conflict on one pair of homologs. The unusual properties of TRKIN may help resolve these conflicts by facilitating a directional switch. ( B ) Meiosis II centromere rotation. Neocentromeres in meiosis I leave knobs near the nuclear periphery during interphase, already prepositioned to move toward the basal cell of the meiotic tetrad (see A). The meiotic spindle forms by a self-organization mechanism . Microtubules are stabilized around chromatin where RanGTP (orange) concentration is highest, and the spindle grows out to form poles. We proposed that neocentromeres move with the forming spindle and rotate linked centromeres in the same direction before stable kinetochore–microtubule contacts are made ( ; ). The fact that TR-1 neocentromeres move early and fast may facilitate this event.

Article Snippet: An E. coli codon-optimized version of the Trkin CDS was synthesized by Genscript and cloned into the expression vector pET-17b (Novagen) modified to include N-terminal 6xHis and GFP tags for affinity protein purification.

Techniques: Concentration Assay